Fluorescent labels and labeled species and their use in analytical elements and determinations

ABSTRACT

Fluorescent labels comprise a polysaccharide bound to a polymeric particle which contains a fluorescent rare earth chelate. These labels can be attached to any of a variety of physiologically reactive species to provide labeled species which have improved stability in aqueous solutions. The labeled species are particularly useful in specific binding assays to determine an immunologically reactive ligand, e.g. a hapten, in either solution or dry analytical techniques.

FIELD OF THE INVENTION

This invention relates to fluorescent labels and to fluorescent labeledphysiologically reactive species useful in biomedical studies andclinical chemistry determinations. These labels and labeled species areparticularly useful in specific binding assays, e.g. immunoassays, todetermine a specific binding ligand, such as a hapten, in humanbiological fluids.

BACKGROUND OF THE INVENTION

In the fields of medicine and clinical chemistry, many studies anddeterminations of physiologically reactive species, e.g. cells, protein,enzymes, cofactors, nucleic acids, substrates, antigens, antibodies,etc. are carried out using "labels" which facilitate the detection orseparation of the materials under observation at low concentration. Inone such application, the diagnosis of pathological conditions and thedetection of drugs or narcotics in humans and animals is often carriedout using labeled materials in specific binding assays using competitivebinding principles.

Whenever labels are used, sensitivity is of prime importance due to thegenerally low levels of biological species that are measured. Procedurescarried out using radiometric labels generally do not have sufficientsensitivity for many low level analytes. In addition, radiometric labelssuffer from the drawbacks of short useful life and handling hazards.

Labeling with magnetic iron oxide has also been proposed, as describedin U.S. Pat. No. 4,452,773 (issued June 5, 1984 to Molday). Thesensitivity of such labels is also limited, and their use in labelingbiological species requires expensive equipment and tedious procedures.

Fluorescent spectroscopy, one of the most sensitive and versatile of theoptical analytical techniques, has become increasingly popular in recentyears to overcome drawbacks of other labeling techniques. Influorescence spectroscopy, a sample containing a fluorescent species isirradiated with light of known spectral distribution within theexitation spectrum of the target fluorescent species. The intensity ofthe resulting characteristic emission spectrum of the fluorescent targetmolecules is determined and is related to the number of target moleculesin the sample. Fluorescent spectroscopy is used extensively for studiesof protein structure, bacterial cell wall reactions and conformationalchanges in enzymes, as well as for determinations of an immunologicallyreactive ligand in a specific binding assay.

Fluorescent labels comprising chelates of a rare earth elementincorporated into polymeric particles of a loadable latex are describedin U.S. Pat. Nos. 4,259,313 (issued Mar. 31, 1981 to Frank et al) andrelated 4,283,382 (issued Aug. 11, 1981 to Frank et al). These labelsexhibit improved efficiency in fluorescence and are particularly usefulfor immunoassays. The polymeric particles serve as carriers forimmunologically reactive species directly attached thereto.

Our colleagues, J. R. Schaeffer, T. J. Chen and M. A. Schen, havediscovered that certain polymers provide exceptionally stablefluorescent labels. These labels are the subject of copending andcommonly assigned U.S. Ser. No. 713,202 filed Mar. 18, 1985 and entitledSTABILIZED FLUORESCENT RARE EARTH LABELS AND LABELED PHYSIOLOGICALLYREACTIVE SPECIES. However, these polymers comprise a small class ofmaterials composed of recurring units derived from certain combinationof monomers.

Although the labels of the Frank et al references represent abreakthrough in clinical chemistry because of their improvedfluorescence efficiency, there is a need to render the broad class ofpolymeric labels described therein more stable in aqueous solutions. Thelabels of Frank et al generally tend to agglutinate spontaneously and tosettle out of solution. They therefore have a shortened storage life.They also demonstrate a tendency to agglutinate prematurely during anassay. The improvements discovered by Schaeffer et al are limited to asmall class of polymeric labels. Hence, it is desirable to havefluorescent labels which avoid the above problems.

SUMMARY OF THE INVENTION

We have discovered novel labels and labeled species having improvedstability in aqueous solutions (e.g. biological fluids). These materialsdo not prematurely agglomerate, can be stored for long periods of timeand require minimal resuspension prior to use. These materials areparticularly useful in specific binding assays, but they can also beused in a variety of biomedical studies where labeling of anyphysiologically reactive species is desired. These labels exhibit thedesirably high sensitivity which accompanies the use of fluorescencespectroscopy.

The unexpected and significantly improved properties of the materials ofthis invention are achieved by the use of a polysaccharide arm to link alabeled polymeric particle with a physiologically reactive species. Thispolysaccharide arm also enhances the sensitivity of the assay toanalytes which might otherwise be undetectable because they are dwarfedby the much larger polymeric particle.

Therefore, in accordance with this invention, a fluorescent labelcomprises a polysaccharide bound to a polymeric particle which isderived from a loadable latex having a discontinuous phase whichconsists essentially of a polymer prepared from one or moreethylenically unsaturated polymerizable monomers, and an aqueous phase.The polymeric particle has incorporated therein a fluorescent rare earthchelate.

This invention also provides a fluorescent labeled physiologicallyreactive species. This labeled species comprises a conjugate of aphysiologically reactive species bound to a polysaccharide, whichconjugate is bound to the polymeric particle described above through thepolysaccharide. In preferred embodiments, this labeled species is alabeled immunologically reactive ligand analog.

Further, this invention provides a dry analytical element whichcomprises an absorbent carrier material and the fluorescent labeledphysiologically reactive species described above. In preferredembodiments, the absorbent material is a porous spreading zone carriedon a support.

This invention also provides a method of labeling a physiologicallyreactive species comprising attaching the species to the fluorescentlabel described above.

Still further, this invention comprises a method for the determinationof an immunologically reactive ligand in an aqueous liquid. The methodcomprises the steps of:

A. in the presence of a receptor for the ligand, contacting a sample ofthe liquid with a fluorescent labeled immunologically reactive ligandanalog as described above,

to form a complex between the receptor and the ligand analog, and

B. fluorometrically detecting the ligand analog.

DETAILED DESCRIPTION OF THE INVENTION

The fluorescent labels of this invention can be used as probes (alsoknown as labels) for a variety of biomedical studies and clinicalchemistry determinations. They can be used to label cells or otherphysiologically reactive species including proteins, nucleic acids (e.g.DNA), enzymes and their substrates, cofactors, viruses, leukocytes,growth factors, antigens, haptens or drugs, antibodies, metabolites,hormones, plant lectins, toxins, radioisotopes and other pharmacalogicalagents and their receptors, and other binding substances enabling thedetection of such substances. Examples of these substances are extensiveand well known in the art.

The labels are particularly useful in specific binding assays todetermine an analyte (i.e. immunologically reactive species). In theseassays, the species to be determined is attached to the label and thelabeled species is placed in competition with unlabeled species forreaction with a common reactant. The species to be determined isreferred to herein as the ligand, and the labeled species as the ligandanalog. Compounds which specifically recognize the ligand and ligandanalog and react to form complexes with them are referred to herein asreceptors.

In performing one such assay, the ligand is placed in competition withthe ligand analog for binding to the receptor. Unknown concentrations ofthe ligand are inferred from the measured signal of the labeled ligandanalog. The reaction proceeds as follows:

ligand+labeled ligand analog+receptor⃡ligand-receptor+labeled ligandanalog-receptor.

In preferred embodiments of this invention, the ligand is an antigen orantibody, the labeled ligand analog is a labeled antigen or antibody andthe specific binding assay is an immunoassay. In the followingdiscussion and presentation of examples, reference will be madeprimarily to these preferred embodiments, but it is to be understoodthat the scope of the invention is inclusive of any other specificbinding assay.

The labels of this invention comprise a polysaccharide bound to a latexpolymeric particle which contain a rare earth chelate. Thepolysaccharide is bound to the particle by any suitable means, e.g.covalent bonding or absorption, but preferably, covalent bonding isused.

Polysaccharides useful in the practice of this invention arecarbohydrates containing at least 3 simple sugar molecules and include,but are not limited to, crosslinked polysaccharides, a polysaccharidederivative which can be hydrolyzed by an endohydrolase,lipopolysaccharides (polysaccharides containing fatty acids), celluloseand cellulosic derivatives, carboxymethylated polysaccharides, inulinand the like. The polysaccharides can be obtained from natural orsynthetic sources. Particularly useful polysaccharides are genericallyknown as dextrans and lipopolysaccharides each having a molecular weightof at least about 1000, and preferably from about 10⁴ to about 10⁷.Lipopolysaccharides are described in, for example, an article by Tsumitaet al, J. Exp. Med., 119, pp. 1017-1025 (1964). Dextrans arepolysaccharides containing a backbone of D-glucose units linkedpredominantly α-D (1-6). Representative dextrans are described in thearticles by Jeanes et al, J.A.C.S., 76, pp. 5041-5052 (1954) and Bankertet al, J. Immun., 123(6), pp. 2466-2474 (1979). Many dextrans arecommercially available. Dextrans are most preferred in the practice ofthis invention. The polysaccharides have functional groups (e.g. hydroxygroups) which are reactive, or can be rendered reactive, with proteinsor other biological molecules.

The polysaccharide is bound to the surface of latex polymer particleswhich contain a rare earth chelate. These polymer particles are preparedwith loadable latices which are described in detail in U.S. Pat. Nos.4,259,313 and 4,283,382 (noted above). Preferably, these labels are"aqueous-stabilized" meaning that the fluorescence of the chelate is notquenched in an aqueous environment.

In general, any fluorescent rare earth chelate which demonstratesfluorescent behavior is useful in the practice of this invention. Inparticular, the chelate comprises a rare earth metal (i.e. a lanthanidemetal) such as europium or terbium. Europium is most preferred.

The chelate also includes a suitable chelating agent. Particularlyuseful chelating agents include 1,3-diketones (e.g. acetylacetonate,p-benzoylacetonate, p-benzoylbenzoate, trifluoro-2-furylacetylacetone,etc.), phthalates, naphthoates (e.g. dinaphthoylmethide, etc.),dipyridines (e.g. 2,2'-bypyridine-1,1'-dioxide,4,4'-dimethyl-2,2'-dipyridine, etc.), terpyridines (e.g.2,2',6',2"-terpyridine, etc.) and phenanthrolines (e.g. o-phenanthrolineisothiocyanate, etc.). Other chelating agents are known to those skilledin the art. The 1,3-diketones are preferred.

The details of "loading" the latices useful in this invention are givenin the Frank et al patents noted above. Generally, the chelate isincorporated in the polymer particles by gradually increasing thehydrophilicity of a solution of a hydrophobe in a water-miscible solventin the presence of uncoagulated, undissolved loadable polymeric latexparticles to a point at which substantially no hydrophobe remainsdissolved in the watermiscible solvent. Up to about 7.5% (based onpolymer weight) of chelate can be "loaded" or imbibed into the polymerparticles in this manner. The concentration of chelate in the polymerparticles can be varied to some extent depending upon the particular useof the label intended. The preparation of a fluorescent label of thisinvention is described in Example 1 below.

Loadable polymer latices useful herein are those which include apolymeric discontinuous phase (particles ) which consists essentially ofone or more polymers prepared from one or more ethylenically unsaturatedpolymerizable monomers, and an aqueous continuous phase. The polymerparticles of these latices generally have an average diameter of fromabout 0.01 to about 2 μm, and preferably from about 0.1 to about 0.5 μm.

Particularly useful latices are those comprising polymers comprising

(a) from 0 to 100 weight percent of units derived from one or more vinylaromatic monomers (i.e. substituted or unsubstituted styrenes and vinylnaphthyls),

(b) from 0 to about 90 weight percent of units derived from one or moreethylenically unsaturated monomers of the structure: ##STR1## wherein Ris hydrogen or substituted or unsubstituted alkyl, preferably of 1 to 5carbon atoms (e.g. methyl, ethyl, chloromethyl, hydroxymethyl,isopropyl, etc.), R₁ is hydrogen or methyl, and R₂ is hydrogen, halogen(e.g. chloro, bromo, etc.), substituted or unsubstituted alkyl,preferably of 1 to 4 carbon atoms (e.g. methyl, hydroxymethyl, ethyl,isopropyl, butyl, etc.), cyano, ##STR2## wherein R₃ is hydrogen or R₄,and R₄ is a substituted or unsubstituted aliphatic group preferablycontaining from 1 to 10 carbon atoms, and optionally one or more oxygenether or nitrogen amido atoms in the chain (e.g. alkyl,alkylene-oxy-alkyl, alkylene-carbonyloxyalkyl, alkylene-carbonamido,hydroxyalkyl, etc.), optionally substituted with one or more amino(primary, secondary, tertiary or quaternary) groups, and

(c) from 0 to 10 weight percent of units derived from one or moreethylenically unsaturated monomers containing one or more sulfonic acidor carboxy groups, or an ammonium or alkali metal (e.g. sodium,potassium, etc.) salt thereof.

Representative monomers of group (a) include styrene, α-methylstyrene,p-bromostyrene, styrene sulfonic acid, sodium salt, vinyltoluene,2-vinylmesitylene, chloroethylsulfonyl methyl styrene,1-vinylnaphthylene, and others known in the art. Representative monomersof group (b) include acrylamide, methacrylamide, n-butyl acrylate,methyl methacrylate, acrylonitrite, propyl acrylate, 2-hydroxyethylacrylate, 2-hydroxypropyl methacrylate, N-isopropylacrylamide, vinylchloride, vinylbromide, and others known in the art. Representativemonomers of group (c) include acrylic acid, methacrylic acid,2-acrylamido-2-methylpropane sulfonic acid, sodium salt,3-methacryloyloxypropane-1-sulfonic acid, sodium salt,2-methacrylamido-2,2-dimethylpropane sulfonic acid, sodium salt,2-(methacryloyloxy)ethyltrimethylammonium methosulfate, vinyl sulfonicacid, potassium salt, and others known to one skilled in the art.

Preferred polymers are composed of from about 40 to 100 weight percentof units derived from a styrene monomer (substituted or unsubstituted asdescribed hereinabove), and from 0 to about 60 weight percent of unitsderived from monomers (b) above wherein R is hydrogen, R₁ is hydrogen ormethyl, and R₂ is cyano, ##STR3## wherein R₃ is hydrogen or R₄ and R₄ issubstituted or unsubstituted alkyl, preferably of 1 to 10 carbon atoms(e.g. methyl, hydroxymethyl, ethyl, isopropyl, t-butyl, hexyl, etc.).

In addition to the units of preferred monomers described above, thepolymers can comprise units derived from one or more ethylenicallyunsaturated monomers other than those of (a), (b), or (c) describedabove. Such monomers are known to one skilled in polymer chemistry.

Particularly useful polymers which can contain a label and be attachedto a polysaccharide are taken from the specific class of polymersdescribed in copending and commonly assigned U.S. patent applicationSer. No. 713,202 of Schaeffer et al, noted above.

The loadable polymer latices useful in preparing the fluorescent labelscan be prepared using well known emulsion polymerization techniques.Generally, they are prepared using free radical initiated reactions ofthe monomers dispersed in an aqueous medium with one or more appropriatesurfactants.

The fluorescent label can be prepared by mixing the polysaccharide and apolymer latex containing the rare earth chelate for a suitable time tofacilitate absorption of the polysaccharide on the surface of thepolymer particles. To covalently bond the polysaccharide to the polymerparticles, either one or both components may be chemically modified tofacilitate reaction of polysaccharide reaction sites with correspondingreaction sites on the polymer particle surfaces.

The fluorescent labeled physiologically reactive species of thisinvention can be prepared in either of two ways. One way is to react thespecies with the fluorescent label described above to covalently bondthe species to the polysaccharide. This procedure may require chemicalmodification of either the fluorescent label (i.e. the polysaccharidelinking arm of the label) or the species, or both.

Alternatively, the physiologically reactive species and polysaccharidecan be reacted to form a polysaccharide-species conjugate before thepolysaccharide is attached to the polymeric particles containing thechelate. Either or both reactants may be modified in order to promotethe reaction. For example, some or all hydroxyl groups of thepolysaccharide may be oxidized to provide pendant aldehyde groups. Thespecies, for example, may contain or be modified to contain amine groupswhich can react with the aldehyde groups of the polysaccharide to form aSchiff base bonding the polysaccharide to the species. Details ofpreparing a representative conjugate with polysaccharides and thyroxineare described below in Examples 1 and 3.

Once having prepared a polysaccharide-species conjugate, it can beattached to the surface of the polymeric particles through thepolysaccharide linking arm to form a labeled physiologically reactivespecies by any suitable technique known to one skilled in the art. Forexample, the conjugate can be absorbed to the polymer particles, orcovalently bonded thereto. Generally, the conjugate and latex are mixedtogether for a suitable time (e.g. up to 72 hours) to facilitateabsorption of conjugate on the surface of the polymer particles. Detailsof the preparation of a representative labeled species are provided inExamples 1 and 3 below.

The fluorescent labeled specific binding ligand analog of this inventioncan be used in specific binding immunoassays, particularly those whichutilize temporal resolution of the specific detecting signal todistinguish it from background. In this immunoassay, a sample of testaqueous liquid is excited in an intermittent fashion and information isaccepted only during the dark cycle when the long-lived fluorescentlabel is still emitting strongly but when other sources of fluorescencehave decayed. Discontinuous excitation can be achieved in a variety ofways, including pulsed laser, mechanical chopping or a continuousexcitation beam, moving the sample in and out of the excitation beam,etc. In general, fluorescent immunoassay techniques are known in theart.

In the practice of this invention, the labeled ligand analog indicatesthe amount of unknown ligand in the test sample. Either the bound orunbound fraction of labeled ligand analog can be measured.

To accomplish a specific binding assay, physical separation of bound andunbound ligand can be carried out using conventional techniques.

In a solution assay, the fluorescent labeled specific binding ligandanalog is generally present in a concentration of up to about 1, andpreferably from about 0.01 to about 1, mg/dl of solution. The receptorcorresponding to the ligand (or analyte) to be determined is generallypresent in an amount of up to about 1, and preferably from about 10⁻⁶ toabout 1 g/dl of solution. Other materials, e.g. buffers, surfactants,etc. can be included in conventional amounts if desired.

The ligand analog and method of this invention are adaptable to bothsolution and dry element assays. The ligand analog, along with itsreceptor, can be provided as part of a diagnostic test kit for eitherdry or solution assays. For solution assays, the kit components can besupplied as lyophilized reagents in individual packets havingpredetermined amounts. Alternatively, they can be provided in bottled orotherwise packaged solutions sufficient in size for one or more assays.Other optional reagents can also be supplied in the kit along withsuitable assay utensils or containers for performing the assay. A dryanalytical element (described below) containing a ligand analog can alsobe supplied as part of the diagnostic kit.

Generally, the ligand analog, its corresponding receptor and test samplebelieved to contain a ligand analyte are physically contacted and mixedin a suitable container (e.g. test tube, petrie dish, beaker, cuvette,etc.). The resulting solution can be incubated, if desired, for a time(e.g. 0.5-4 hours) at a temperature of up to about 37° C. to promote theformation of a complex of the receptor with both the ligand analog andthe ligand in the test sample. The sample is then evaluated by measuringthe fluorescence of bound (i.e. complexed) or unbound (i.e.noncomplexed) label. Such an evaluation can be done visually or withsuitable fluorometric detection equipment and procedures.

The method of this invention can also be utilized with a dry analyticalelement which can be composed of an absorbent carrier material, i.e.thin sheet of self-supporting absorbent or bibulous material, such as afilter paper or strip, which contains the labeled physiologicallyreactive species of this invention. Such elements can also contain areceptor for a specific binding assay immobilized in a suitable mannerand kept isolated from the corresponding ligand analog prior to theassay. Such elements are known in the art as test strips, diagnosticelements, dip sticks, diagnostic agents and the like.

When employed in dry analytical elements, the ligand analog of thisinvention can be incorporated into a suitable carrier material byimbibition or impregnation, or can be coated on a suitable absorbentmaterial. Useful carrier materials are insoluble and maintain theirstructural integrity when exposed to water or physiological fluids suchas urine or serum. Useful carrier materials can be prepared from paper,porous particulate structures, cellulose, porous polymeric films, wood,glass fiber, woven and nonwoven fabrics (synthetic and nonsynthetic) andthe like. Useful materials and procedures for making such elements arewell known in the art as exemplified in U.S. Pat. Nos. 3,092,465 (issuedJune 4, 1963 to Adams et al), 3,802,842 (issued Apr. 9, 1974 to Lange etal), 3,915,647 (issued Oct. 28, 1975 to Wright), 3,917,453 (issued Nov.4, 1975 to Milligan et al), 3,936,357 (issued Feb. 3, 1976 to Milliganet al), 4,248,829 (issued Feb. 3, 1981 to Kitajima et al), 4,255,384(issued Mar. 10, 1981 Kitajima et al), and 4,270,920 (issued June 2,1981 to Kondo et al), and U.K. Pat. No. 2,052,057 (published Jan. 21,1981).

Preferably, the dry analytical elements of this invention have at leastone porous spreading zone as the absorbent carrier material. This zonecan be self-supporting (i.e. composed of a material rigid enough tomaintain its integrity), but preferably it is carried on a separatesupporting substrate (commonly called a support). Such a support can beany suitable dimensionally stable, and preferably, transparent (i.e.radiation transmissive) material which transmits electromagneticradiation of a wavelength between about 200 and about 900 nm. A supportof choice for a particular element should be compatible with theintended mode of detection (reflection or transmission spectroscopy).Useful support materials include paper, metal foils, polystyrene,polyesters [e.g. poly(ethylene terephthalate)], polycarbonates,cellulose esters (e.g. cellulose acetate), etc.

The porous spreading zone can be prepared from any suitable fibrous ornon-fibrous material or mixtures of either or both. The void volume andaverage pore size of this zone can be varied depending upon the useintended. For example, if whole blood or other liquid samples containinghigh molecular weight materials are to be assayed, the void volume andaverage pore size are generally greater than if serum or urine is to beassayed.

Useful spreading zones can be prepared using fibrous materials, eithermixed with a suitable binder material or woven into a fabric, asdescribed in U.S. Pat. No. 4,292,272 (issued Sept. 29, 1981 to Kitajimaet al). Alternatively the spreading zone is prepared from polymericcompositions (e.g. blush polymers) or particulate materials, with orwithout binding adhesives, as described in U.S. Pat. Nos. 3,992,158(issued Nov. 16, 1976 to Przybylowicz et al) and 4,258,001 (issued Mar.24, 1981 to Pierce et al). Other useful spreading zone materials aredescribed in W. German OLS No. 3,150,102 (published July 29, 1982) andJapanese Patent Publication 57(1982)-101760 (published June 24, 1982).It is desirable that the spreading zone be isotropically porous, meaningthat the porosity is the same in each direction in the zone as createdby interconnected spaces or pores between particles, fibers, polymericstrands, etc.

The elements can have one or more reagent zones, spreading zones,registration zones, mordant zones, radiation-blocking or filter zones,subbing zones, barrier zones, buffer zones, etc. The zones are generallyin fluid contact with each other meaning that fluids, reagents andreaction products can pass between superposed regions of adjacent zones.Stated in another manner, fluid contact refers to the ability totransport components of a fluid between the zones in fluid contact.Preferably, the zones are separately coated layers, although two or morezones can be a single layer, or a zone can contain two or more separatelayers.

The fluorescent labeled ligand analog of this invention can beincorporated in any zone of the element. Alternatively, it can be addedto the test sample which is subsequently applied to the element, or theligand analog can be separately (either subsequently or simultaneously)added to the element with the test sample. The receptor corresponding tothe ligand to be determined can also be in any zone of the element inimmobilized form, or added to the element simultaneously with the testsample. If both the ligand analog and the receptor are incorporated intothe element prior to the assay, they must be kept isolated from eachother until the assay is carried out.

In the elements of this invention, the coverage of the ligand analog canbe varied widely, but it is generally present in a coverage of up toabout 1, and preferably from about 10⁻⁶ to about 1 g/m². The receptorcan be present in a coverage of up to about 200, and preferably fromabout 40 to about 200 g/m². A variety of other desirable, but optional,reagents and addenda can be present in the element in amounts known toone skilled in the art. Such materials include interactive reagents,surfactants, buffers, binders, pigments, activators, etc.

A variety of different elements, depending on the method of assay, canbe prepared in accordance with the present invention. Elements can beconfigured in a variety of forms, including elongated tapes of anydesired width, sheets, slides or chips.

The assay of this invention can be manual or automated. In general, inusing the dry elements, determination of a ligand is made by taking theelement from a supply roll, chip packet or other source and physicallycontacting it with a sample (e.g. 1-100 μl) of the liquid to be testedin the presence of the receptor. Such contact can be accomplished in anysuitable manner, e.g. dipping or immersing the element into the sampleor, preferably, by spotting the element by hand or machine with a dropof the sample with a suitable dispensing means.

After sample application, the element is exposed to any conditioning,such as incubation, heating or the like, that may be desirable toquicken or otherwise facilitate obtaining any test result. Determinationof the ligand is achieved by measuring the fluorescence of either thebound (i.e. complexed) or unbound (i.e. noncomplexed) labeled ligandanalog.

The following examples are presented to illustrate the practice of thepresent invention. In these examples, the materials were obtained asfollows: N-carbobenzoxyglycylglycine from Sigma Chemical Co. (St. Louis,Mo.), cyclohexylmorpholinoethylene glycol (mol. wt. 750) from AldrichChemical Co. (Milwaukee, Wis.), sodium cyanoborohydride from AlfaProducts (Danvers, Mass.), pellitized carbon black from Cabots ChemicalCo. (Boston, Mass.), poly(vinyl butyral) from Monsanto Co. (St. Louis,Mo.), bovine gamma globulin (BGG) from Miles Laboratories, Inc.(Naperville, Ill.), Zonyl FSN™ surfactant from DuPont (Wilmington,Del.), Staphylococcus aureus particles from Calbiochem-Behring (LaJolla,Calif.), sodium periodate from Fisher Scientific Co. (Rochester, N.Y.),Dextran B1355 from Dr. M. D. Slocki (Biochemistry Research FermentationLaboratories, Peoria, Ill.), Dextran T70, T10 and 500 and Sepharose™2B/6B from Pharmacia Fine Chemicals (Piscataway, N.J.), inulin fromSigma Chemical Co. (St. Louis, Mo.), and the remaining reagents wereeither prepared using conventional procedures or obtained from EastmanOrganic Chemicals (Rochester, N.Y.).

EXAMPLE 1 Europium Labeled-Dextran Ligand Analog for DeterminingThyroxine

A fluorescent labeled ligand analog of the present invention useful forthe determination of the ligand thyroxine was prepared in the followingmanner:

Synthesis of Thyroxine Methyl Ester

Thyroxine (20 g) was suspended in dry methanol (500 ml). The suspensionwas saturated with hydrogen chloride gas, cooled and saturated again.The crystals were collected, washed with methanol and ether, and thendried. The resulting thyroxine methyl ester hydrochloride crystals wereadded to 500 ml of methanol and an equimolar concentration ofconcentrated sodium hydroxide was added with stirring. Deionizeddistilled water (500 ml) was added to this solution. The solution waschilled to recrystallize the thyroxine methyl ester from the solution.The crystals were then collected and air dried.

Synthesis of N-carbobenzoxyglycylglycylthyroxine Methyl Ester

Thyroxine methyl ester (5 g), as prepared above,N-carbobenzoxyglycylglycine (1.67 g) andcyclohexylmorpholinoethylcarbodiimide (5.3 g) were combined in 40 ml ofdimethylacetamide. The mixture was stirred at room temperature for 24hours, poured into deionized water, and the resulting residue collectedand washed with water. The residue was recrystallized from methanol andwater, yielding the product, N-carbobenzoxyglycylglycylthyroxine methylester.

Synthesis of Glycylglycylthyroxine Methyl Ester Hydrobromide

N-carbobenzoxyglycylglycylthyroxine methyl ester (2.3 g), as preparedabove, was suspended in 60 ml of hydrobromic and acetic acids (31% HBrin acetic acid). The mixture was stirred at room temperature for 5hours. The resulting hydrobromide salt was collected by precipitationwith ether, filtered, washed with ether and dried in vacuo to yieldglycylglycylthyroxine methyl ester hydrobromide.

Oxidation of Dextran Polysaccharides

Several dextran polysaccharides were oxidized in preparation forcovalently bonding the thyroxine component thereto.

A. Dextran B1355 was oxidized by the following procedure: 2 g of thepolysaccharide was dissolved in 200 ml deionized, distilled water. Solidsodium periodate (0.43 g) was added and the pH was adjusted to 6.0. Thereaction was stirred at room temperature for 1 hour and then dialyzedextensively against distilled running water. The product shown below wasobtained by lyophilization. ##STR4##

B. Dextran T-70 (4 g) was oxidized as described in part A above with thefollowing exceptions: 4 g of Dextran T-70 was dissolved in 80 ml ofdeionized distilled water and 0.86 g of sodium periodate was added.

C. Dextran T-10 (4 g) was oxidized as described in part B above.

Synthesis of Thyroxine Methyl Ester Diglycyl Dextran Conjugates

Thyroxine methyl ester diglycyl polysaccharide conjugates were preparedby reduction of the Schiff base formed between the amine of thyroxinemethyl ester glycylglycine and the aldehydes of the oxidized dextranswith sodium cyanoborohydride. Reaction conditions were designed toobtain various thyroxine-to-polysaccharide ratios.

Oxidized Dextran B1355 (0.114 g) was dissolved in 10 ml of 0.2 molarpotassium phosphate buffer, pH 8.0. Diglycylthyroxine methyl esterhydrobromide (0.034 g) was dissolved in 2 ml of dimethylacetamide andwas then added to the polysaccharide solution, followed by the additionof 0.06 g of sodium cyanoborohydride. The reaction was stirred at roomtemperature for 2-3 days. The solution was then dialyzed extensivelyagainst 25% dimethyl formamide in water, followed by dialysis againstdistilled water and lyophilization to obtain the product.

The concentration of thyroxine in the resulting thyroxine-polysaccharideconjugate was determined spectrophotometrically at 325 nm against aknown concentration of thyroxine-polysaccharide conjugate with anextinction coefficient of 6300. From this data, the unknownthyroxine-topolysaccharide ratio was calculated.

Conjugates using the oxidized Dextrans T-70 and T-10 were similarlyprepared.

Formation of Ligand Analogs

The diglycyl thyroxine methyl esterpolysaccharide conjugates describedabove were adsorbed onto the surface of various labeled polymer latexparticles in loaded latices to form ligand analogs. Varioushapten-polysaccharide conjugate to latex ratios were used in thesepreparations. The preparation of diglycyl thyroxine methylesterpolysaccharide-europium (Eu⁺³) chelate-loadedpoly(styrene-co-acrylamide-co-methacrylic acid) (weight ratio 85/10/5)summarized below is representative. Diglycyl thyroxine methylester-Dextran B1355 (22 mg) (thyroxine:Dextran ratio of 10,881:1) wassuspended in 3 ml of deionized distilled water. An amount of the latexequivalent to 1 ml of 8% solids, was added dropwise with stirring to thesuspension. The reaction was stirred at room temperature for 2-5 daysand was then chromatographed on a Sepharose™ 2B/68 column. The latexband was collected and assayed for percent solids and iodine content.The hapten-polysaccharide conjugate to latex ratio, which ranged from1.4:1 to 4.4:1, was determined from these results.

The method described above was used to Prepare the following labeledligand analogs using the indicated latices and thyroxine-polysaccharideconjugates.

Poly(styrene-co-acrylamide-co-methacrylic acid) (Eu⁺³ chelate) (weightratio 85/10/5) was sensitized with the following conjugates:

diglycyl thyroxine methyl ester-Dextran B1355 (6889:1thyroxine:polysaccharide ratio)

diglycyl thyroxine methyl ester-Dextran T-70

diglycyl thyroxine methyl ester-Dextran T-10

In addition, the following latex copolymers were sensitized withdiglycyl thyroxine methyl ester-Dextran B1355 (10,881:1thyroxine:polysaccharide ratio).

poly(styrene-co-methacrylamide-co-methacrylic acid) (weight ratio85/10/5) (Eu⁺³ chelate)

poly(styrene-co-methacrylic acid) (weight ratio 95/5) (Eu⁺³ chelate)

polystyrene (Eu⁺³ chelate)

poly(styrene-co-acrylamide) (weight ratio 90/10) (Eu⁺³ chelate)

poly(hydroxymethylstyrene-co-acrylamide-comethacrylic acid) (weightratio 85/10/5) (Eu⁺³ chelate)

poly[styrene-co-acrylamide-co-2-(2-methacryloyloxyethylamino)-4,6-dichloro-5-triazine](weight ratio 88/10/2) (Eu⁺³ chelate)

EXAMPLE 2 Evaluation of labeled Thyroxine Analog

An evaluation of a thyroxine analog was carried out in solution in thefollowing manner using the labeled ligand analog diglycyl thyroxinemethyl ester-Dextran B1355-poly(styrene-co-acrylamide-comethacrylicacid) (weight ratio 85/10/5) (Eu⁺³ chelate) prepared as described inExample 1 hereinabove.

This ligand analog was evaluated as a label for a thyroxine assay byconsidering the following factors:

(1) The ability of the ligand analog to specifically bind toantithyroxine receptor.

(2) The ability of the specific binding with the receptor to be reversedin the presence of free thyroxine.

(3) Nonspecific binding of the ligand analog to antibodies not receptorsfor thyroxine.

The labeled thyroxine analog noted above was diluted to 0.01125% solidsin glycine acetate buffer (pH 7) and combined with serially diluted hightiter thyroxine antiserum in glass microculture tubes. Antiserumdilutions ranged from 1:6 to 1:192. After overnight incubation at roomtemperature, the tubes were evaluated under ultraviolet light foragglutination. Agglutination was observed with antiserum dilutions up toand including the 1:192 dilution. This demonstrated the first factornoted above.

The reversal of the analog-antibody binding in the presence of freethyroxine was examined by adding free thyroxine (3.3×10⁻⁶ molar finalconcentration) to the assay described above. No agglutination of thelabeled latex was observed indicating that free thyroxine caused theanalog-antibody binding to be reversed. This demonstrates the secondfactor noted above.

The thyroxine analog was also incubated with serial dilutions ofantibovine gamma globulin (anti-BGG) which is not a receptor forthyroxine. No agglutination was observed demonstrating the third factornoted above.

EXAMPLE 3 Europium Labeled-Lipopolysaccharide Thyroxine Analog

A thyroxine analog useful for the determination of thyroxine wasprepared in the following manner:

Synthesis of Thyroxine Polyethyleneglycol Ester Hydrochloride

L-Thyroxine (5 g) was suspended in 40 g of moltenmonomethoxypolyethyleneglycol (mol. wt. 750) and gently heated on a hotplate while saturating with HCl. Stirring was continued for anadditional hour and the solution again saturated with HCl. After 2hours, water (200 ml) was added and the precipitate collected andthoroughly washed with cold water and freeze dried to give the productwhich was used in the following preparatory steps.

Preparation of Fatty Acid-Polysaccharide (Lipopolysaccharide)

Polysaccharides were acrylated by reaction with fatty acid chlorides inpyridine as described by Tsumita et al in J. Exp. Med., 119, pp.1017-1025 (1964). The preparation of palmitoyl-polysaccharide whichfollows is representative.

Dextran 70 (10 g) was suspended in 100 ml of pyridine. Ten ml ofpalmitoyl chloride was added dropwise with stirring. After stirring for72 hours at room temperature, the mixture was poured into isopropanol,washed with isopropanol and ether, and dried. The product yield was 11.5g.

A similar procedure was used to prepare the following:

Palmitoyl-Inulin

Palmitoyl-Dextran 500

Palmitoyl-Dextran B1355

Oxidation of the various Dextran and inulin lipopolysaccharides wasaccomplished according to the procedure described for the oxidation ofthe polysaccharides in Example 1 hereinabove.

Synthesis of Palmitoyl-Polysaccharide-Thyroxine Ester

Esterified thyroxine was covalently bound to the oxidizedlipopolysaccharides by the formation and subsequent reduction in thepresence of sodium cyanoborohydride of a Schiff base. The followingmethod of preparation of palmitoyl-Dextran 70-diglycylthyroxine methylester is representative.

Oxidized palmitoyl-Dextran 70 (0.5 g), as prepared above, was dissolvedin 50 ml of 0.2 molar potassium phosphate buffer (pH 8.0).Diglycylthyroxine methyl ester hydrobromide, from above (150 mg), in 10ml dimethylacetamide was added, followed by the addition of 100 mgsodium cyanoborohydride. The mixture was stirred for 18 hours at roomtemperature, dialyzed against 25% N,N-dimethylformamide in water andlyophilized to yield the product.

Palmitoyl-inulin-diglycylthyroxine methyl ester and palmitoyl-DextranB1355-diglycylthyroxine methyl ester were similarly prepared. Bothconjugates were prepared from the hydrobromide salts of thyroxineesters.

A similar procedure was used for the production of conjugates made fromhydrochloride salts of thyroxine esters, such as diglycylthyroxinemethyl ester hydrochloride and thyroxine-polyethylene glycol esterhydrochloride, with the following exceptions: the oxidizedlipopolysaccharides were dissolved in 0.1 molar potassium phosphatebuffer (pH 7.0), the ester was dissolved in N,N-dimethylformamide, andfollowing stirring the product was collected by precipitation frommethanol, thoroughly washed with N,N-dimethylformamide, redissolved anddialyzed with water and lyophilized. Using this procedure, the followingconjugates were prepared: palmitoyl-Dextran 70-thyroxine polyethyleneglycol ester and palmitoyl-Dextran 500-thyroxine diglycyl andpolyethylene glycol esters.

Thyroxine ester-oleyl polysaccharide conjugates were also prepared.

Oleyl-Polysaccharide Preparation

Carboxymethylated Dextran 70-thyroxine methyl ester (thyroxine:Dextranratio of 9:1) and carboxymethyl cellulose were acylated with oleic acidchloride using a similar procedure as above with the exception thatfollowing incubation with stirring the product was washed withisopropanol and acetone before drying. In addition, theoleyl-carboxymethyl cellulose preparation was stirred for only 4 hourswith gentle heating.

Oleyl-Carboxymethyl Cellulose-Diglycylthyroxine Methyl Ester Preparation

Oleyl-carboxymethyl cellulose (0.5 g) was dissolved in 10 ml ofdistilled water. Cyclohexylmorpholinoethylcarbodiimidemetho-p-toluenesulfonate (0.5 g) and diglycylthyroxine methyl esterhydrobromide (0.3 g), in 10 ml dimethylacetamide, were added. Themixture was stirred for 18 hours at room temperature. The product wasprecipitated by the addition of methanol, washed thoroughly insuccession with methanol, N,N-dimethylformamide, and methanol, dialyzedagainst distilled water, and lyophilized.

General Procedure for Preparation of a Ligand Analog

A fatty acid-polysaccharide-thyroxine conjugate (50 mg) was added to 5ml of potassium phosphate buffer (0.01-0.05 molar, pH 6-8). A suitablelabeled latex (5-8% solids) was then added dropwise with stirring to theconjugate solution. The final weight ratio of conjugate to latex solidswas in the range of 1:5 to 1:2. The resulting suspensions were allowedto stir at room temperature for 24 hours and then purified bychromatography on Bio Gel A-5 or Sepharose 2B/6B supports.

Table I hereinbelow lists the conjugate and labeled latex components ofthe thyroxine analogs so prepared.

                  TABLE I                                                         ______________________________________                                        Conjugate          Polymer Latex                                              ______________________________________                                        Palmitoyl-Dextran 70-                                                                            Poly( .sub.--m, -p-vinyltoluene)                           diglycyl thyroxine (Eu.sup.+3 chelate)                                        methyl ester                                                                  Palmitoyl-Dextran 70-                                                                            Polystyrene (Eu.sup.+3                                     diglycyl thyroxine chelate)                                                   methyl ester                                                                  Palmitoyl-Dextran 70-                                                                            Poly(styrene-co-acryl                                      diglycyl thyroxine amide-co-methacrylic                                       methyl ester       acid) (weight ratio                                                           85/10/5) (E.sup.+3                                                            chelate)                                                   *Palmitoyl-Dextran 70-                                                        thyroxine polyethylene                                                        glycol ester                                                                  *Palmitoyl-Dextran 500-                                                       diglycyl thyroxine                                                            methyl ester                                                                  *Palmitoyl-Dextran 500-                                                       thyroxine polyethylene                                                        glycol ester                                                                  *Palmitoyl-Dextran B1355-                                                     diglycyl thyroxine                                                            methyl ester                                                                  Palmitoyl-Dextran 500-                                                                           Poly( .sub.--m, -p-vinyltoluene)                           thyroxine polyethylene                                                                           (Eu.sup.+3 chelate)                                        glycol ester                                                                  Oleyl-carboxymethylated                                                                          Poly( .sub.--m, -p-vinyltoluene)                           Dextran 70-thyroxine                                                                             (Eu.sup.+3 chelate)                                        methyl ester                                                                  Oleyl-carboxymethyl                                                                              Polystyrene (Eu.sup.+3                                     cellulose-diglycyl chelate)                                                   thyroxine methyl ester                                                        Oleyl-hydroxypropyl-                                                                             Polystyrene (Eu.sup.+3                                     carboxymethyl cellulose-                                                                         chelate)                                                   diglycyl thyroxine methyl                                                     ester                                                                         Palmitoyl-inulin-diglycyl                                                                        Polystyrene (Eu.sup.+3                                     thyroxine methyl ester                                                                           chelate)                                                   Palmitoyl-inulin-diglycyl                                                                        Poly( .sub.--m, -p-vinyltoluene)                           thyroxine methyl ester                                                                           (Eu.sup.+3 chelate                                         Palmitoyl-inulin-diglycyl                                                                        Poly(styrene-co-acryl-                                     thyroxine methyl ester                                                                           amide-co-methacrylic                                                          acid) (weight ratio                                                           85/10/5) (Eu.sup.+3                                                           chelate)                                                   ______________________________________                                         *Each of these conjugates was individually adsorbed on labeled latex          particles of both poly(.sub.--m,-pvinyltoluene) (Eu.sup.+3 chelate) and       poly(styreneco-acrylamide-co-methacrylic acid) (weight ratio 85/10/5)         (Eu.sup.+3 chelate).                                                     

EXAMPLE 4 Evaluation of Labeled Thyroxine Analog

An evaluation of a labeled thyroxine analog was carried out in solutionin the following manner using the ligand analog palmitoyl-DextranB1355-diglycyl thyroxine methyl esterpoly(styrene-co-acrylamide-co-methacrylic acid) (weight ratio 85/10/5)(Eu⁺³ chelate) prepared as described in Example 3 hereinabove.

This evaluation was carried out in a fashion comparable to theevaluation described in Example 2. Specific binding of the thyroxineanalog to the antithyroxine receptor was obtained up to and including a1:96 dilution of the antiserum. This binding was completely reversed inthe presence of free thyroxine. No specific binding of the analog toanti-bovine gamma globulin was observed indicating the usefulness ofthis analog for thyroxine immunoassay.

EXAMPLE 5 Solution Immunoassay for Phenytoin (Dilantin) Synthesis of5,5-Diphenylhydantoin-3-(4-Butylamine Acetate)

5,5-Diphenylhydantoin, sodium salt (10 g) and 4-bromobutylnitrile (5.4g) were combined in 75 ml N,N-dimethylformamide. The reaction mixturewas stirred under nitrogen at 80°-90° C. for 1 hour. TheN,N-dimethylformamide was removed under vacuum, and the product(5,5-diphenylhydantoin-3-butylnitrile) was recrystallized frommethanol/acetonitrile and was hydrogenated using PtO₂ as the catalyst inglacial acetic acid under nitrogen. After the hydrogenation, thecatalyst was removed by filtration through a course sintered glassfunnel containing celite. The solvent was then removed by evaporationand the resulting crude product was recrystallized from methanol/ethylacetate.

Synthesis of a Phenytoin-butylamine Polysaccharide Conjugate

A conjugate was synthesized from 5,5-diphenylhydantoin-3-(4-butylamineacetate) and Dextran T-70 by the procedure described in Example 1 forthe synthesis of the thyroxine-Dextran conjugates.

Preparation and Use of Labeled Phenytoin Analog

The conjugates described above were absorbed onto the surface of Eu⁺³-labeled poly(styrene-co-acrylamide-co-methacrylic acid) (weight ratio85/10/5) latex particles by the procedure described in Example 1.

The resulting phenytoin analog was evaluated in an immunoassay in thefollowing manner. Antiphenytoin antibody immobilized on Staphylococcusaureus particles (5 mg) in 50 μl buffer (0.01 molar potassium phosphate,0.15 molar NaCl, pH 7.2) and increasing levels of phenytoin (2×10⁻⁹molar to 2×10⁻⁴ molar, 100 μl) were dispensed into small tubes.Phenytoin analog (1:1000 dilution) (50 μl) was added to each tube. Thetube contents were then mixed, incubated at room temperature for 30minutes and centrifuged. The supernatent (100 μl) was removed from eachtube and combined with 900 μl of water in a polystyrene microabsorbancecell and the resulting fluorescence was measured. As shown in thefollowing table, the fluorescence generally increased with increasingphenytoin levels greater than 1 nanomolar.

    ______________________________________                                        Phenytoin (nanomolar)                                                                         Fluorescence (nano A)                                         ______________________________________                                        0               0.30                                                          1               0.29                                                          10              0.38                                                          100             0.57                                                          1000            0.92                                                          10,000          1.0                                                           100,000         1.0                                                           ______________________________________                                    

EXAMPLE 6 Dry Analytical Element and its Use in Immunoassay for BovineGamma Globulin

A dry element was used to assay for bovine gamma globulin (BGG) in thefollowing manner.

A lipopolysaccharide obtained by oxidizing palmitoyl Dextran B1355 in10⁻² molar periodate, followed by dialysis and recovery bylyophilization, was absorbed directly onto the surface of Eu⁺³chelate-containing latex particles ofpoly(styrene-co-acrylamide-co-methacrylic acid) (weight ratio 85/10/5)to form a fluorescent label. For the performance of a fluorescentimmunoassay agglutination test, BGG antigen was incubated with thefluorescent label in the presence of a selective reducing agent, sodiumcyanoborohydride. The resulting ligand analog was purified by gelchromatography on agarose to remove any noncovalently bound BGG.

The activity of the labeled analog was demonstrated using a dry testelement having the following format and components:

    ______________________________________                                                               Range g/m.sup.2                                        ______________________________________                                        Polystyrene beads having anti-BGG anti-                                                                 40-200                                              body adsorbed thereto                                                         Poly(n-butylacrylate-co-styrene-co-                                                                      1-15                                               2-acrylamido-2-methylpropane sulfonic                                         acid) (weight ratio 70/20/10) adhesive                                        Zonyl FSN ™ surfactant                                                                              0.1-5                                                Potassium chloride       0.1-2                                                Boric acid               0.1-2                                                Pellitized carbon black  0.1-10                                               Poly(vinyl butyral)      0.1-10                                               Poly(ethylene terephthalate)                                                  Support                                                                       ______________________________________                                    

Mixtures of labeled BGG at 10⁻⁸ molar and unlabeled BGG at 10⁻⁹ -10⁻⁵molar were prepared. A 10 μl sample of each mixture was spotted onto asample of the element. All element samples were incubated for 1-3minutes at 37° C., after which a phosphate buffer (pH 7.5) saline washsolution was applied to remove unbound BGG. A dose response curvegenerated by measuring the bound fluorescent label in the elementindicated that as unlabeled BGG concentration increased, the amount ofbound labeled BGG decreased.

EXAMPLE 7 Dry Analytical Element and Its Use in Immunoassay forThyroxine

An element was prepared similar to that described in Example 6 exceptthat anti-thyroxine antibody was immobilized on the polystyrene beads inplace of anti-BGG. A fluorescent labeled thyroxin analog (10⁻¹⁰ molar)as described in Example 1 and unlabeled thyroxine samples (10⁻⁹ -10⁻⁶molar) were applied to samples of the element by the procedure describedin Example 6. After a one minute incubation, the element samples werewashed with a phosphate buffer (pH 7) saline solution to remove unboundthyroxine. The presence of bound labeled thyroxine remaining in theelement was evaluated by measure of the fluorescence. A dose responsecurve generated from the data indicated that the higher the unboundthyroxine concentration in the test sample, the lower the amount ofbound labeled thyroxine in the element.

EXAMPLE 8 Stability Comparison

This example illustrates the improved stability of the immunoreagentcompositions of this invention by comparing them to similar compositionstaught in the art, namely those taught in U.S. Pat. No. 4,283,382 (notedhereinabove). This comparison was carried out in the following manner.

Fluorescent labels (Eu⁺³ chelate) were prepared according to theteaching of U.S. Pat. No. 4,283,382 using poly(styrene-co-methacrylicacid) (weight ratio 95/5) (latex 1) and poly(styrene-co-acrylamide)(weight ratio 90/10) (latex 2). Thyroxine methyl ester glycyl glycylDextran B conjugate was absorbed to the polymeric particles of a portionof each Eu⁺³ -containing latex noted above at a ratio of 1.2:1 (latex 1)and 0.8:1 (latex 2), respectively, to form thyroxine analogs. All fourpreparations were stored at 10° C. for several months after which theywere evaluated for stability.

Large clumps of precipitated latex were observed in both preparations offluorescent labels prepared according to the prior art whilesubstantially no precipitated latex was observed in the preparationscontaining thyroxine analog.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

We claim:
 1. A fluorescent labeled physiologically reactive speciescomprisinga conjugate of a physiologically reactive species bound to apolysaccharide, said conjugate bound through said polysaccharide to apolymeric particle which is derived from a loadable latex having adiscontinuous phase which consists essentially of a polymer preparedfrom one or more ethylenically unsaturated polymerizable monomers, andan aqueous continuous phase, said polymeric particle having incorporatedtherein a fluorescent rare earth chelate.
 2. The labeled species ofclaim 1 wherein said physiologically reactive species is a specificbinding species.
 3. The labeled species of claim 1 wherein saidpolysaccharide is a dextran or a lipopolysaccharide and has a molecularweight of from about 10⁴ to about 10⁷ daltons.
 4. The labeled species ofclaim 1 wherein the chelate is a chelate of europium or terbium and a1,3-diketone, phthalate, naphthoate, dipyridine, terpyridine, orphenanthroline chelating agent, andsaid polymer comprises(a) from 0 to100 weight percent of units derived from a substituted or unsubstitutedstyrene or vinyl naphthyl, (b) from 0 to about 90 weight percent ofunits derived from an ethylenically unsaturated monomer of the structure##STR5## wherein R is hydrogen or alkyl, R₁ is hydrogen or methyl, andR₂ is hydrogen, halogen, alkyl, cyano, ##STR6## wherein R₃ is hydrogenor R₄, and R₄ is an aliphatic group, and (c) from 0 to about 10 weightpercent of units derived from an ethylenically unsaturated monomercontaining one or more sulfonic acid or carboxy groups, or an ammoniumor alkali metal salt thereof.
 5. The labeled species of claim 4 whereinsaid unit of part (a) is styrene or a styrene drivative and said unit ofpart (b) has the recited structure wherein R is hydrogen, R₁ is hydrogenor methyl and R₂ is cyano, ##STR7## wherein R₃ is hydrogen or alkyl andR₄ is alkyl.
 6. A fluorescent labeled specific binding ligand analog fordetermining a specific binding ligand in an aqueous liquid, said labeledanalog comprisinga conjugate of said ligand bound to a polysaccharide,said conjugate bound through said polysaccharide to a polymeric particlewhich is derived from a loadable latex having a polymeric discontinuousphase which consists essentially of a polymer comprising(a) from 0 to100 weight percent of units derived from a substituted or unsubstitutedstyrene or vinyl naphthyl, (b) from 0 to about 90 weight percent ofunits derived from an ethylenically unsaturated monomer of the structure##STR8## wherein R is hydrogen or alkyl, R₁ is hydrogen or methyl and R₂is hydrogen, halogen, alkyl, cyano, ##STR9## wherein R₃ is hydrogen orR₄, and R₄ is an aliphatic group, and (c) from 0 to about 10 weightpercent of units derived from an ethylenically unsaturated monomercontaining one or more sulfonic acid or carboxy groups, or an ammoniumor alkali metal salt thereof, and an aqueous continuous phase, saidpolymeric particle having incorporated therein a fluorescent rare earthchelate.
 7. The labeled analog of claim 6 wherein said specific bindingligand is a hapten.
 8. A dry analytical element comprising an absorbentcarrier material, and a fluorescent labeled physiologically reactivespecies,said labeled species comprising a conjugate of a physiologicallyreactive species bound to a polysaccharide, said conjugate bound throughsaid polysaccharide to a polymeric particle which is derived from aloadable latex having a discontinuous phase which consists essentiallyof a polymer prepared from one or more ethylenically unsaturatedpolymerizable monomers, and an aqueous continuous phase, said polymericparticle having incorporated therein a fluorescent rare earth chelate.9. The element of claim 8 wherein said species is a specific bindingligand, and said polysaccharide is a dextran or a lipopolysaccharide andhas a molecular weight of from about 10⁴ to about 10⁷ daltons.
 10. A dryanalytical element for the determination of a specific binding ligand inan immunoassay, said element comprisinga support having thereon a porousspreading zone, and a fluorescent labeled ligand analog of said ligand,said labeled analog comprising a conjugate of said ligand bound to apolysaccharide, said conjugate bound through said polysaccharide to apolymeric particle which is derived from a loadable latex having adiscontinuous phase which consists essentially of a polymer, and anaqueous continuous phase, said polymer comprising(a) from 40 to 100weight percent of units derived from a substituted or unsubstitutedstyrene or vinyl naphthyl, and (b) 0 to about 60 weight percent of unitsderived from an ethylenically unsaturated monomer of the structure##STR10## wherein R is hydrogen or alkyl, R₁ is hydrogen or methyl, andR₂ is hydrogen, halogen, alkyl, cyano, ##STR11## wherein R₃ is hydrogenor R₄, and R₄ is an aliphatic group, said polymeric particle havingincorporated therein a fluorescent rare earth chelate.
 11. The elementof claim 10 wherein said rare earth metal is europium or terbium, saidunit of part (a) is styrene or a styrene derivative and said unit ofpart (b) has the recited structure wherein R is hydrogen, R₁ is hydrogenor methyl and R₂ is cyano, ##STR12## wherein R₃ is hydrogen or alkyl andR₄ is alkyl.
 12. The element of claim 10 wherein said ligand is ahapten.
 13. The element of claim 10 further comprising an immobilizedreceptor for said ligand.
 14. A method for the determination of aspecific binding ligand in an aqueous liquid, said method comprising thesteps of:A. in the presence of a receptor for said ligand, contacting asample of said liquid with a fluorescent labeled specific binding ligandanalog,said ligand analog comprising a conjugate of said ligand bound toa polysaccharide, said conjugate bound through said polysaccharide to apolymeric particle which is derived from a loadable latex having adiscontinuous phase which consists essentially of a polymer preparedfrom one or more ethylenically unsaturated polymerizable monomers, andan aqueous continuous phase, said polymeric particle having incorporatedtherein a fluorescent rare earth chelate to form a complex between saidreceptor and said ligand analog, B. separating said complex fromuncomplexed ligand analog, and C. fluorometrically detecting either saidcomplexed or uncomplexed ligand analog as a measure of said ligand. 15.The method of claim 14 wherein said ligand is a hapten.
 16. The methodof claim 14 wherein said polysaccharide is a dextran or alipopolysaccharide and has a molecular weight of from about 10⁴ to about10⁷ daltons, and said polymer comprises(a) from 0 to 100 weight percentof units derived from a substituted or unsubstituted styrene or vinylnaphthyl, (b) from 0 to about 90 weight percent of units derived from anethylenically unsaturated monomer of the structure ##STR13## wherein Ris hydrogen or alkyl, R₁ is hydrogen or methyl, and R₂ is hydrogen,halogen, alkyl, cyano, ##STR14## wherein R₃ is hydrogen or R₄, and R₄ isan aliphatic group, and (c) from 0 to about 10 weight percent of unitsderived from an ethylenically unsaturated monomer containing one or moresulfonic acid or carboxy groups, or an ammonium or alkali metal saltthereof.
 17. The method of claim 14 wherein said complexed ligand analogis detected.
 18. A dignostic test kit for the determination of aspecific binding ligand, said kit comprising(i) a receptor for saidligand and (ii) a fluorescent labeled specific binding ligand analogcomprisinga conjugate of said ligand bound to a polysaccharide, saidconjugate bound through said polysaccharide to a polymeric particlewhich is derived from a loadable latex having a discontinuous phasewhich consists essentially of a polymer prepared from one or moreethylenically unsaturated polymerizable monomers, and an aqueouscontinuous phase, said polymeric particle having incorporated therein afluorescent rare earth chelate.